Thrombin Activatable Fibrinolysis Inhibitor, TAFI, is a 60 kDa glycoprotein found in human plasma. It is also known as procarboxypeptidase B, carboxypeptidase B, plasma carboxypeptidase B, carboxypeptidase U and carboxypeptidase R. It plays an intrinsic part in the blood coagulation process during which it is transformed into an activated form, TAFIa, whereupon it acts upon the fibrin matrix which comprises a developing blood clot to prevent its dissolution. Imbalances in the blood coagulation process are thought to be the origin of a large and disparate number of disease conditions which are linked by an unwanted build up of fibrin. The scale of fibrin build up is determined by the delicate equilibrium between two biochemical cascades in the human body; the coagulation and fibrinolysis cascades. These cascades are an integral part of maintaining hemostasis.
To maintain hemostasis in the blood, mammals have developed mechanisms to repair the body in the event of vascular injury. The injured blood vessel will constrict to reduce the blood flow to the area. Platelets will aggregate to reduce the loss of blood from the area followed by fibrinogen which polymerizes and forms a fibrin clot. This clot will cover the area of vascular damage preventing blood loss. Once the blood vessel has been repaired the clot will then dissolve. The coagulation cascade is responsible for the forming of a clot; the fibrinolysis cascade is responsible for the dissolution of the clot.
Studies have shown that these two processes are intrinsically linked through the generation of α-thrombin. α-Thrombin is the final product of the blood coagulation cascade and is responsible for the conversion of soluble plasma fibrinogen to an insoluble fibrin matrix. Polymerized fibrin provides a haemostatic plug which prevents blood loss from the site of vascular injury and provides a provisional matrix which enhances the subsequent repair process. In addition to mediating coagulation, α-thrombin also reduces the rate at which blood clots are broken down by the serine protease plasmin. The anti-fibrinolytic activity of α-thrombin results from its activation of TAFI. TAFI circulates in normal plasma at a concentration of about 75 nM in an inactive form. Thrombin converts the inactive zymogen to the active TAFI (TAFIa); a reaction that is augmented about 1250-fold by thrombomodulin. Once activated, TAFIa cleaves both C-terminal arginine and lysine residues from the developing fibrin clot. The removal of di-basic amino acids from the surface of the fibrin matrix attenuates clot lysis by inhibiting the binding of the key mediators of fibrinolysis: tissue plasminogen activator (tPA) and its substrate, plasminogen, which is the precursor of plasmin. Both tPA and plasminogen contain a structural motif called a kringle domain which binds tightly to C-terminal lysine residues. The removal of these binding sites prevents the formation of a ternary complex between tPA, plasminogen and fibrin and this inhibits the conversion of plasminogen to plasmin thus protecting the clot from rapid degradation.
It can be seen that if the equilibrium between coagulation and fibrinolysis is in favor of coagulation, then there will be a larger amount of fibrin present than normal. This makes it more likely that the subject will develop one or more of the conditions in which thrombus build up is implicated. By the use of a TAFIa inhibitor, TAFIa will not be able to act upon a developing fibrin clot as described above to inhibit-fibrinolysis of the clot. Therefore, a TAFIa inibitor should serve to enhance the fibrinolysis cascade.
The use of TAFI inhibitors to treat certain conditions is known in the art. Whilst the use of TAFIa inhibitors to treat such conditions is unknown, certain weak, non-specific TAFIa inhibitors have been identified.
U.S. Pat. No. 5,993,815 teaches the use of a peptide that binds to the TAFI zymogen, inhibiting activation of the TAFI zymogen, to treat those disorders where a C-terminal lysine or arginine is cleaved from an intact peptide. The disorders include arthritis, sepsis, thrombosis, strokes, deep vein thrombosis and myocardial infarctions. The peptide used is an antibody or a functionally active fragment. The peptide should be used in an amount to promote fibrinolysis in vivo.
McKay et al, Biochemistry, 17, 401 (1978), discloses the testing of a number of compounds as competitive inhibitors of bovine carboxypeptidase B of pancreatic origin. Inhibition was measured by the inhibitor's efficiency in protecting the active centre tyrosine and glutamic acid of bovine carboxypeptidase B from irreversible alkylation by bromoacetyl-D-arginine or bromoacetamidobutylguanidine. It is suggested that such inhibitors could act as bradykinin potentiators.
Bovine enzymes of pancreatic origin are very different to those found in human plasma, so one would not expect inhibitors of one to inhibit the other. Moreover, such inhibitors are directed towards a very different utility. Accordingly the above reference contains no teaching of TAFIa inhibitors or their utility.
Redlitz et al, J. Clin. Invest., 96, 2534 (1995), teaches the involvement of plasma carboxypeptidase B (pCPB, or TAFI) in the formation of clots. The lysis of blood clots was followed in the absence and presence of pCPB, whereupon it was found that the presence of pCPB slowed clot lysis. To confirm that pCPB was responsible, two control reactions were run: (1) where the lysis experiment was repeated in the presence of pCPB and a carboxypeptidase inhibitor, PCI; and (2) where the lysis reaction was conducted in the presence of plasma from which pCPB was removed. In both cases lysis proceeded uninhibited.
Boffa et al, J. Biol. Chem., 273, 2127 (1998), compares plasma and recombinant TAFI and TAFIa with respect to glycosylation, activation, thermal stability and enzymatic properties. Inhibition constants for three competitive inhibitors were determined: ε-aminocaproic acid (ε-ACA), 2-guanidinoethyl-mercaptosuccinic acid (GEMSA) and potato carboxypeptidase inhibitor (PCI).
There are large numbers of carboxypeptidases characterized by cleaving the C-terminal amino acid from a peptide. They may be divided into acidic, neutral or basic, depending on the type of amino acid they cleave. Basic carboxypeptidases cleave arginine, lysine and histidine. TAFIa is a specific subset of basic carboxypeptidases. In terms of the present invention, the inhibitors disclosed above by Redlitz, et al. and Boffa, et al., are too weak, non-specific or otherwise unsuitable to be considered as suitable TAFIa inhibitors for therapeutic application. Further, while the role of TAFIa in clot lysis is explained, there is no suggestion that TAFIa inhibitors can be used to treat disease.
PCT publication WO00/66550 discusses a broad class of compounds useful as inhibitors of carboxypeptidase U. Inhibitors of carboxypeptidase U are postulated to facilitate fibrinolysis and thus the compounds are taught as useful in the treatment of thrombotic conditions. Although details of a suitable assay are given, there is no data to support this assertion.
PCT publication WO00/66152 discloses formulations containing a carboxypeptidase U inhibitor and a thrombin inhibitor. Suitable carboxypeptidase U inhibitors are those of PCT publication WO00/66550. The formulations are taught as being useful primarily in treating thrombotic conditions.